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Binding of ω-conotoxin to receptor sites associated with the voltage-sensitive calcium channel
Neuroscience Letters, 71 (1986) 203-208
203
Elsevier Scientific Publishers Ireland Ltd.
NSL 04226
Binding of co-conotoxin to receptor sites associated with the voltage-sensitive calcium channel T. Abe l, K. Koyano I, H. Saisu 1, Y. N i s h i u c h i 2 a n d S. S a k a k i b a r a 2 1Department of Neurochemistry, Brain Research Institute, Niigata University, 1 Asahimachi, Niigata 951, and 2Peptide Institute, Mino-shi, Osaka 562 (Japan) (Received 7 July 1986; Revised version received 21 July 1986; Accepted 23 July 1986)
Key wordy: t~J-Conotoxin Calcium channel Synaptic membrane The binding of radioiodinated to-conotoxin GVIA, a probable Ca channel antagonist, to synaptic plasma membranes of rat brain was examined. Two kinds of specific binding sites were found with apparent dissociation constants of 10 pM and 0.5 nM and maximum binding capacities of 0.5 and 3.4 pmol/mg prot., respectively. The binding of the toxin was not affected by high concentrations of Ca antagonists or an agonist, indicating distinct binding sites of the toxin from those of these drugs. Divalent and trivalent metal ions strongly inhibited the binding. The order of their inhibitory potencies was similar to that for inhibition of the Ca current through certain Ca channels. These results suggest that the binding sites of ctJ-conotoxin GVIA are functionally related to the Ca2+-binding site postulated to be in the pore of the Ca channel.
Ca plays a key role in many physiological processes, such as muscle contraction, cell growth, nerve spikes and secretion of hormones and neurotransmitters [2]. In the nervous system, a major route for Ca influx into cells is the voltage-sensitive Ca channel. In order to understand the molecular mechanism underlying Ca influx, it is necessary to isolate the molecule constituting the voltage-sensitive Ca channel. Recently, dihydropyridine Ca antagonists and agonists have been used extensively as probes of the Ca channel [4, 18]. However, these drugs usually have little or no influence on Ca influx and transmitter release induced by depolarization of presynaptic terminals (cf. ref. 10). Moreover, patch-clamp studies have revealed the existence of Ca channels that are insensitive to a dihydropyridine Ca agonist [14]. Thus, other probes besides these dihydropyridines are required for identification of the voltagesensitive Ca channel, og-Conotoxin GVIA (¢o-CgTX), a neurotoxic peptide from the venom of a marine snail, Conus geographus [15], seems to be a suitable probe: it blocks the release of transmitter from nerve terminals, probably by arresting Ca influx through the voltage-sensitive Ca channel [9]. This toxin is probably also active Correspondence." T. Abe, Department of Neurochemistry, Brain Research Institute, Niigata University, Asahimachi 1, Niigata 951, Japan. 0304-3940/86/$ 03.50 © 1986 Elsevier Scientific Publishers Ireland Ltd.
204
~6
~4 X
I
0.4
018
125"['-,,,-CoTX,
1.2
16
nM
10
2.0
30
Bound, pmol/mg
Fig. I. Binding of e~-~-~I-CgTX to SPM. Typical results are shown. A: total binding (O) and non-specific binding ( ) . B: Scatchard plot of specific binding defined as the difference between total and non-specific binding. The data points obtained from A were fitted to a curve using the LIGAND program [I I].
in the brain, as it causes a tremor when injected into the brain [13, 15]. We report here the specific binding of the toxin to brain membranes. ¢o-CgTX was synthesized as described previously [I 3]. Synthetic o~-CgTX was iodihated with Na-J25I using chloramine T, and monoiodinated ¢o-CgTX ((~-12sl-CgTX) was separated from unlabeled toxin on CM-Sephadex (T.A. et al., to be published). The toxicity of ¢o-t25I-CgTX to goldfish [13] was comparable with that of the native toxin. Binding experiments were carried out at 4°C. The mixture (total volume, 0.5 ml) was incubated for 90 min in 20 mM Tris-HCl, pH 7.5, containing synaptic plasma membranes (SPM) prepared from rat forebrain [8] (1 2 Izg prot.), 0.1% bovine serum albumin (BSA) and various concentrations of ~o-12SI-CgTX (2100 or 210 Ci/ 100 -d 90
"~ 80 x I-.-
7e 3 I
H m 60
5(]
1'o Time
. h
Fig. 2. Time course of dissociation of the tu-t2~l-CgTX SPM complex at 4'C. After incubation with 0.2 (O) or 0.02 nM (©) e~-~2sI-CgTX for 90 min, dissociation was initiated by the addition of 1 ,uM unlabeled toxin. Aliquots were taken at the indicated times. Non-specific binding, determined by taking aliquots from the samples incubated with e~-~2~I-CgTX in the presence of 1 lzM unlabeled eJ-CgTX, was subtracted from total binding.
205
mmol). The binding was terminated by rapid filtration of 0.40-ml aliquots through cellulose acetate membranes (Sartorius: pore size, 0.2/tm) under reduced pressure followed by 3 washings with 5 ml of 20 mM Tris-HCl~).15 M NaClq2.1% BSA, pH 7.5 (Solution A). Just before filtration, 25 pl of 20 mM Tris-HC1-3 M NaC1, pH 7,5, was added to each sample to reduce non-specific binding of co-125I-CgTX to SPM and filters. Before use, filters were soaked in solution A for 1 h. Non-specific binding was measured in the presence of 1 pM unlabeled og-CgTX. All binding assays were carried out in triplicate and mean values are shown. Fig. IA shows an example of the total and non-specific binding of co-125I-CgTX to SPM. A Scatchard plot of the specific binding defined as the difference between the total and non-specific binding could not be fitted to a straight line (Fig. ! B), indicating multiple binding sites. When analyzed using an Apple II version (MED-58) of the L I G A N D program [11], the data indicated the presence of two types of binding sites. From 4 experiments, the following binding parameters were obtained: apparent Kdl (dissociation constant for site 1), 10.5 ___3.1 pM (mean ___S.D.); apparent Kd2 (for site 2), 0.52+0.35 nM; Bmaxl (maximum number of binding sites for site 1), 0.52+0.04 pmol/mg prot.; nmax2 ( f o r site 2), 3.4+ 1.6 pmol/mg prot.; B m a x l / B m a x 2 , 0.20 + 0.09. The dissociation of ~o-125I-CgTX from sites 1 and 2 were both very slow, as shown in Fig. 2. The addition of 1 mM EDTA and 1 mM EGTA to the binding medium did not increase the specific binding. The binding of ~-125I-CgTX (0.2 nM) was inhibited by metal ions (Fig. 3). The binding of 0.02 nM ~o-125I-CgTX was inhibited very similarly (data not shown), indicating that the bindings of the toxin to sites I and 2 were both sensitive to metal ions. While Na ÷ and K ÷ were weak inhibitors (IC50 about 50 mM), divalent metal ions inhibited the binding very effectively. Among the metal ions -m"
100 -4
x
--0
o
g
50
¢..p 8 -
0
1:5
! |w
1
8
6
4
2
-log (C0nc.). M Fig. 3. Inhibition of og-tesI-CgTX binding by metal ions. Before addition of og-12sI-CgTX (0.2 nM), SPM was incubated with various metal ions (chloride salts) for 10 min. A: effects o f monovalent and divalent metal ions. L,, Na+; II, K+; A, Sr2+; (3, Ba2+; O , Ca 2+. B: effects of divalent and trivalent metal ions. (3, Mg 2 ' ; O, C o 2 ~; &, Ni 2 +; D, Cd 2 ~; II, La 3+. Values are expressed as percentage of binding in the absence of metal ions. The experiments with La 3+ and Cd 2+ were carried out in 20 m M PIPES-NaOH, pH 6.5, containing 0.05% BSA, to avoid precipitation of proteins. Other metal ions were less inhibitory at pH 6.5 than at pH 7.5.
206 (Ca 2+ , Sr 2+ , Ba 2+) that can c a r r y large a m o u n t s o f c u r r e n t t h r o u g h the voltage-sensitive Ca channel [6], only Ca 2+ was a strong inhibitor: it was i n h i b i t o r y at as low a c o n c e n t r a t i o n as 3 0 / t M . A m o n g the metal ions tested, La 3+ was by far the strongest inhibitor. The IC50 o f La 3+ was less than I # M , a n d the b i n d i n g was c o m p l e t e l y inhibited by 0.1 m M La 3+, as shown in Fig. 3B. The inhibition by Co 2+ and Ni 2+ were a p p r e c i a b l e , but that by M g 2+ was weak. The o r d e r o f the i n h i b i t o r y potencies o f metal ions was: La 3+ > C d 2+ > C a 2+ > Ni 2+ = C o 2+ > Ba 2+ > Sr 2 ÷ > M g 2+ > Na + =K ÷" N e i t h e r toxins acting on the N a channel n o r those acting on the K channels affected the b i n d i n g o f ~o-125I-CgTX to S P M (Table I). M o r e i m p o r t a n t l y , the b i n d i n g o f ~-125I-CgTX (0.02 a n d 0.2 n M ) was n o t significantly influenced by any o f the 3 classes o f drugs s u p p o s e d to act on the voltage-sensitive Ca channel [l 7] even at m u c h higher c o n c e n t r a t i o n s than n o r m a l l y used. F o r instance, n i t r e n d i p i n e binds to its receptor in the brain m e m b r a n e s with a Kd o f a b o u t 0.14).2 n M (e.g. see ref. 5), but at 40 p M it did n o t affect the b i n d i n g o f m J z 5 I - C g T X . N i t r e n d i p i n e binding to brain m e m b r a n e s requires divalent metal ions such as C a 2+ a n d Sr 2+ [5]. H o w e v e r , d i h y d r o p y r i d i n e d r u g s did n o t affect the b i n d i n g o f o~-CgTX even in the presence o f Ca 2+ (Table I). The present study d e m o n s t r a t e d the presence o f specific b i n d i n g sites for o~-CgTX
TABLE 1 CHEMICALS HAVING NO SIGNIFICANT INFLUENCE ON THE BINDING OF (o-~>I-CgTX (CAUSING LESS THAN 10% DECREASE OR INCREASE) SPM was incubated wilh the indicated chemicals for 1 h, before addition of 0.02 or 0.2 nM ~-I>I-CgTX. Incubation with dihydropyridineswas carried out in the dark. Chemicals
Concentration
Target
Apamin Tetraethylammonium 4-Aminopyridine 3.4-Diaminopyridine
I/tM 5 mM 5 mM 5 mM
K channels
Tetrodoloxin Veratridine ;'-Toxin from T i t y u s ~
I ILM 0.1 mM I izM
Na channel
Bay K8644 Nitrcndipine Nifedipine Nisoldipine_+0.1 mM CaCI2 Verapamil D-600 Diltiazem Pimozide
40 ,uM 40/tM 40 ,uM 40/~M 0. I mM 0.1 mM 0.1 mM 40/tM
Ca channel
"Prcpared by Dr. J.R. Giglio, Universidade de Sao Paulo.
207
in SPM. Probably og-CgTX attacks the voltage-sensitive Ca channel or an entity closely associated with it [9]. Thus, the specific binding sites of to-CgTX revealed in this study are very likely components of the voltage-sensitive Ca channel or molecules associated with it. Two high-affinity binding sites were detected. It is unknown whether these two binding sites correspond to the different types of Ca channel in the nervous system demonstrated by patch-clamp studies [14]. The dissociations of oJ-CgTX from sites 1 and 2 were both very slow, suggesting that the action of the toxin in the brain may be slowly reversible or irreversible as in the peripheral nervous system [9]. The binding of og-125I-CgTX was not affected by either Ca antagonists or agonist. Ca antagonists other than dihydropyridines (see Table I) bind to sites allosterically linked to the binding site of dihydropyridines [16]. The present findings indicate that ~-CgTX binds to different sites from the binding sites of any of these drugs. Further studies are required to determine whether og-CgTX and these drugs bind to distinct sites on the same molecule or to different molecules. The binding of ~-~25I-CgTX was strongly inhibited by low concentrations of divalent and trivalent metal ions. The order of the inhibitory potencies of metal ions on the binding of oJ-CgTX was very similar to that on the Ca current through the Ca channel in neuroblastoma cells that shows slow inactivation [19] and the Ca channel involved in transmitter release in nerve terminals in the brain [12]. This suggests that ~-CgTX interacts with sites related to the site of action of these metal ions in the Ca channel. Recent models [1, 7] postulate that the selective permeability of Ca 2+ through the Ca channel at high concentrations of Na ÷ and K ÷ in physiological media is achieved by preferential, high-affinity binding of two Ca ions to sites located in the pore of the channel. Divalent and trivalent metal ions may bind to the same sites, but be very poorly translocated through the channel, except in the case of Sr 2+ and Ba 2+ [6]. Thus, ~o-CgTX may bind to sites located close to the Ca2+-binding sites or allosterically related to them. After the completion of this work, the binding of to-CgTX to chick brain synaptosomes was reported [3]. Although the binding of t~-CgTX is not characterized in detail, the results are in agreement with this study in that usual Ca antagonists do not affect the binding of to-CgTX. The properties of the binding of to-CgTX reported here indicate that to-CgTX should be a useful probe for investigating the structure and function of the voltagesensitive Ca channel. We thank Dr. M. Satake for encouragement and helpful discussion. Thanks are also due to Dr. M. Okuda, Department of Psychiatry, for analysis of binding data using the L I G A N D program, kindly provided by the Biomedical Computing Technology Information Center, Nashville, TN, U.S.A. We are grateful to the drug companies (Bayer, Knoll, Tanabe and Janssen) for the supply of Ca antagonists and an agonist. This study was supported by a Grant-in-Aid (to T.A.) for Special Project Research on the Molecular Mechanisms of Bioelectrical Responses, from the Japanese Ministry of Education, Science and Culture.
208 I Almers, W. and McCleskey, E.W., Non-selective conductance in calcium channels of frog muscle: calcium selectivity in a single-file pore, J. Physiol. (London), 353 (1984) 585--608. 2 Campbell, A.K., Intracellular Calcium. Its Universal Role as Regulator, John Wiley & Sons, Chichester, 1983. 3 Cruz, J. and Olivera, B.M., Calcium channel antagonists. ~n-Conotoxin defines a new high affinity site, J. Biol. Chem., 261 (1986) 6230~6233. 4 Glossmann, H.. Ferry, D.R., L/ibbecke, F., Mewes, R. and Hofmann, F., Calcium channels: direct identification with radioligand binding studies, TIPS, 3 (1982) 431 437. 5 Gould, R.J., Murphy, K.M.M. and Snyder, S.H. [3H]Nitrendipinc-labeled calcium channels discriminate inorganic calcium agonists and antagonists, Proc. Natl. Acad. Sci. USA, 79 (1982) 3656 3660. 6 ttagiwara, S. and Byerly, L., Calcium channel, Annu. Rev. Neurosci., 4 (1981) 69 125. 7 Hess, P. and Tsien, R.W., Mechanism of ion permeation through calcium channels, Nature (London) 309 (1984) 453 456. 8 Jones, D.H. and Matus, A.I., Isolation of synaptic plasma membrane from brain by combined riotation-sedimentation density gradient centrifugation, Biochim. Biophys. Acta, 356 (1974) 276 287. 9 Kerr. L.M. and Yoshikami, D., A venom peptide with a novel presynaptic blocking action, Naturc (London), 308 (1984) 282 284. 10 Miller, R.J., How many types of calcium channels exist in neurones?, TINS, 8 (1985) 45 47. I 1 Munson, P.J. and Rodbard, D., LIGAND: a versatile computerized approach for characterization of ligand-binding system, Anal. Biochem., 107 (1980) 220 239. 12 Nachshen, D.A., Selectivity of the Ca binding sitc in synaptosome Ca channels, J. Gen. Physiol., 83 (1984) 941 967. I3 Nishiuchi, Y., Kumagaye, K., Noda, Y., Watanabe, T.X. and Sakakibara, S., Synthesis and secondary structure determination of ~v-eonotoxin GVIA: a 27-peptide with three intramolecular disulfide bonds, Biopolymers, in press. 14 Nowycky, M.C., Fox, A.P. and Tsien, R.W., Three types of neuronal calcium channel with different calcium agonist sensitivity, Nature (London), 316 (1985) 440 443. 15 Olivera, B.M., Mclntosh, J.M., Cruz, L.J., Luque, F.A. and Gray, W.R., Purification and sequence of a prcsynaptic peptidc toxin from Conus geographus venom, Biochcmistry, 23 (1984) 5087 5090. 16 Schramm, M. and Towert, R., Modulation of calcium channel function by drugs, Life Sci., 37 (I 985j 1843 1861). 17 Spedding, M., Calcium antagonist subgroup, TIPS, 6 (1985) 109 114. 18 Towert. R. and Schramm. M., Recent advances in the pharmacology of the calcium channel, TIPS, 5(1984) 111 113. 19 Tsunoo. A., Yoshii, M. and Narahashi, T., Differential block of two types of calcium channels in neuroblastoma cells, Biophys. J., 47 (1985) 433a.
203
Elsevier Scientific Publishers Ireland Ltd.
NSL 04226
Binding of co-conotoxin to receptor sites associated with the voltage-sensitive calcium channel T. Abe l, K. Koyano I, H. Saisu 1, Y. N i s h i u c h i 2 a n d S. S a k a k i b a r a 2 1Department of Neurochemistry, Brain Research Institute, Niigata University, 1 Asahimachi, Niigata 951, and 2Peptide Institute, Mino-shi, Osaka 562 (Japan) (Received 7 July 1986; Revised version received 21 July 1986; Accepted 23 July 1986)
Key wordy: t~J-Conotoxin Calcium channel Synaptic membrane The binding of radioiodinated to-conotoxin GVIA, a probable Ca channel antagonist, to synaptic plasma membranes of rat brain was examined. Two kinds of specific binding sites were found with apparent dissociation constants of 10 pM and 0.5 nM and maximum binding capacities of 0.5 and 3.4 pmol/mg prot., respectively. The binding of the toxin was not affected by high concentrations of Ca antagonists or an agonist, indicating distinct binding sites of the toxin from those of these drugs. Divalent and trivalent metal ions strongly inhibited the binding. The order of their inhibitory potencies was similar to that for inhibition of the Ca current through certain Ca channels. These results suggest that the binding sites of ctJ-conotoxin GVIA are functionally related to the Ca2+-binding site postulated to be in the pore of the Ca channel.
Ca plays a key role in many physiological processes, such as muscle contraction, cell growth, nerve spikes and secretion of hormones and neurotransmitters [2]. In the nervous system, a major route for Ca influx into cells is the voltage-sensitive Ca channel. In order to understand the molecular mechanism underlying Ca influx, it is necessary to isolate the molecule constituting the voltage-sensitive Ca channel. Recently, dihydropyridine Ca antagonists and agonists have been used extensively as probes of the Ca channel [4, 18]. However, these drugs usually have little or no influence on Ca influx and transmitter release induced by depolarization of presynaptic terminals (cf. ref. 10). Moreover, patch-clamp studies have revealed the existence of Ca channels that are insensitive to a dihydropyridine Ca agonist [14]. Thus, other probes besides these dihydropyridines are required for identification of the voltagesensitive Ca channel, og-Conotoxin GVIA (¢o-CgTX), a neurotoxic peptide from the venom of a marine snail, Conus geographus [15], seems to be a suitable probe: it blocks the release of transmitter from nerve terminals, probably by arresting Ca influx through the voltage-sensitive Ca channel [9]. This toxin is probably also active Correspondence." T. Abe, Department of Neurochemistry, Brain Research Institute, Niigata University, Asahimachi 1, Niigata 951, Japan. 0304-3940/86/$ 03.50 © 1986 Elsevier Scientific Publishers Ireland Ltd.
204
~6
~4 X
I
0.4
018
125"['-,,,-CoTX,
1.2
16
nM
10
2.0
30
Bound, pmol/mg
Fig. I. Binding of e~-~-~I-CgTX to SPM. Typical results are shown. A: total binding (O) and non-specific binding ( ) . B: Scatchard plot of specific binding defined as the difference between total and non-specific binding. The data points obtained from A were fitted to a curve using the LIGAND program [I I].
in the brain, as it causes a tremor when injected into the brain [13, 15]. We report here the specific binding of the toxin to brain membranes. ¢o-CgTX was synthesized as described previously [I 3]. Synthetic o~-CgTX was iodihated with Na-J25I using chloramine T, and monoiodinated ¢o-CgTX ((~-12sl-CgTX) was separated from unlabeled toxin on CM-Sephadex (T.A. et al., to be published). The toxicity of ¢o-t25I-CgTX to goldfish [13] was comparable with that of the native toxin. Binding experiments were carried out at 4°C. The mixture (total volume, 0.5 ml) was incubated for 90 min in 20 mM Tris-HCl, pH 7.5, containing synaptic plasma membranes (SPM) prepared from rat forebrain [8] (1 2 Izg prot.), 0.1% bovine serum albumin (BSA) and various concentrations of ~o-12SI-CgTX (2100 or 210 Ci/ 100 -d 90
"~ 80 x I-.-
7e 3 I
H m 60
5(]
1'o Time
. h
Fig. 2. Time course of dissociation of the tu-t2~l-CgTX SPM complex at 4'C. After incubation with 0.2 (O) or 0.02 nM (©) e~-~2sI-CgTX for 90 min, dissociation was initiated by the addition of 1 ,uM unlabeled toxin. Aliquots were taken at the indicated times. Non-specific binding, determined by taking aliquots from the samples incubated with e~-~2~I-CgTX in the presence of 1 lzM unlabeled eJ-CgTX, was subtracted from total binding.
205
mmol). The binding was terminated by rapid filtration of 0.40-ml aliquots through cellulose acetate membranes (Sartorius: pore size, 0.2/tm) under reduced pressure followed by 3 washings with 5 ml of 20 mM Tris-HCl~).15 M NaClq2.1% BSA, pH 7.5 (Solution A). Just before filtration, 25 pl of 20 mM Tris-HC1-3 M NaC1, pH 7,5, was added to each sample to reduce non-specific binding of co-125I-CgTX to SPM and filters. Before use, filters were soaked in solution A for 1 h. Non-specific binding was measured in the presence of 1 pM unlabeled og-CgTX. All binding assays were carried out in triplicate and mean values are shown. Fig. IA shows an example of the total and non-specific binding of co-125I-CgTX to SPM. A Scatchard plot of the specific binding defined as the difference between the total and non-specific binding could not be fitted to a straight line (Fig. ! B), indicating multiple binding sites. When analyzed using an Apple II version (MED-58) of the L I G A N D program [11], the data indicated the presence of two types of binding sites. From 4 experiments, the following binding parameters were obtained: apparent Kdl (dissociation constant for site 1), 10.5 ___3.1 pM (mean ___S.D.); apparent Kd2 (for site 2), 0.52+0.35 nM; Bmaxl (maximum number of binding sites for site 1), 0.52+0.04 pmol/mg prot.; nmax2 ( f o r site 2), 3.4+ 1.6 pmol/mg prot.; B m a x l / B m a x 2 , 0.20 + 0.09. The dissociation of ~o-125I-CgTX from sites 1 and 2 were both very slow, as shown in Fig. 2. The addition of 1 mM EDTA and 1 mM EGTA to the binding medium did not increase the specific binding. The binding of ~-125I-CgTX (0.2 nM) was inhibited by metal ions (Fig. 3). The binding of 0.02 nM ~o-125I-CgTX was inhibited very similarly (data not shown), indicating that the bindings of the toxin to sites I and 2 were both sensitive to metal ions. While Na ÷ and K ÷ were weak inhibitors (IC50 about 50 mM), divalent metal ions inhibited the binding very effectively. Among the metal ions -m"
100 -4
x
--0
o
g
50
¢..p 8 -
0
1:5
! |w
1
8
6
4
2
-log (C0nc.). M Fig. 3. Inhibition of og-tesI-CgTX binding by metal ions. Before addition of og-12sI-CgTX (0.2 nM), SPM was incubated with various metal ions (chloride salts) for 10 min. A: effects o f monovalent and divalent metal ions. L,, Na+; II, K+; A, Sr2+; (3, Ba2+; O , Ca 2+. B: effects of divalent and trivalent metal ions. (3, Mg 2 ' ; O, C o 2 ~; &, Ni 2 +; D, Cd 2 ~; II, La 3+. Values are expressed as percentage of binding in the absence of metal ions. The experiments with La 3+ and Cd 2+ were carried out in 20 m M PIPES-NaOH, pH 6.5, containing 0.05% BSA, to avoid precipitation of proteins. Other metal ions were less inhibitory at pH 6.5 than at pH 7.5.
206 (Ca 2+ , Sr 2+ , Ba 2+) that can c a r r y large a m o u n t s o f c u r r e n t t h r o u g h the voltage-sensitive Ca channel [6], only Ca 2+ was a strong inhibitor: it was i n h i b i t o r y at as low a c o n c e n t r a t i o n as 3 0 / t M . A m o n g the metal ions tested, La 3+ was by far the strongest inhibitor. The IC50 o f La 3+ was less than I # M , a n d the b i n d i n g was c o m p l e t e l y inhibited by 0.1 m M La 3+, as shown in Fig. 3B. The inhibition by Co 2+ and Ni 2+ were a p p r e c i a b l e , but that by M g 2+ was weak. The o r d e r o f the i n h i b i t o r y potencies o f metal ions was: La 3+ > C d 2+ > C a 2+ > Ni 2+ = C o 2+ > Ba 2+ > Sr 2 ÷ > M g 2+ > Na + =K ÷" N e i t h e r toxins acting on the N a channel n o r those acting on the K channels affected the b i n d i n g o f ~o-125I-CgTX to S P M (Table I). M o r e i m p o r t a n t l y , the b i n d i n g o f ~-125I-CgTX (0.02 a n d 0.2 n M ) was n o t significantly influenced by any o f the 3 classes o f drugs s u p p o s e d to act on the voltage-sensitive Ca channel [l 7] even at m u c h higher c o n c e n t r a t i o n s than n o r m a l l y used. F o r instance, n i t r e n d i p i n e binds to its receptor in the brain m e m b r a n e s with a Kd o f a b o u t 0.14).2 n M (e.g. see ref. 5), but at 40 p M it did n o t affect the b i n d i n g o f m J z 5 I - C g T X . N i t r e n d i p i n e binding to brain m e m b r a n e s requires divalent metal ions such as C a 2+ a n d Sr 2+ [5]. H o w e v e r , d i h y d r o p y r i d i n e d r u g s did n o t affect the b i n d i n g o f o~-CgTX even in the presence o f Ca 2+ (Table I). The present study d e m o n s t r a t e d the presence o f specific b i n d i n g sites for o~-CgTX
TABLE 1 CHEMICALS HAVING NO SIGNIFICANT INFLUENCE ON THE BINDING OF (o-~>I-CgTX (CAUSING LESS THAN 10% DECREASE OR INCREASE) SPM was incubated wilh the indicated chemicals for 1 h, before addition of 0.02 or 0.2 nM ~-I>I-CgTX. Incubation with dihydropyridineswas carried out in the dark. Chemicals
Concentration
Target
Apamin Tetraethylammonium 4-Aminopyridine 3.4-Diaminopyridine
I/tM 5 mM 5 mM 5 mM
K channels
Tetrodoloxin Veratridine ;'-Toxin from T i t y u s ~
I ILM 0.1 mM I izM
Na channel
Bay K8644 Nitrcndipine Nifedipine Nisoldipine_+0.1 mM CaCI2 Verapamil D-600 Diltiazem Pimozide
40 ,uM 40/tM 40 ,uM 40/~M 0. I mM 0.1 mM 0.1 mM 40/tM
Ca channel
"Prcpared by Dr. J.R. Giglio, Universidade de Sao Paulo.
207
in SPM. Probably og-CgTX attacks the voltage-sensitive Ca channel or an entity closely associated with it [9]. Thus, the specific binding sites of to-CgTX revealed in this study are very likely components of the voltage-sensitive Ca channel or molecules associated with it. Two high-affinity binding sites were detected. It is unknown whether these two binding sites correspond to the different types of Ca channel in the nervous system demonstrated by patch-clamp studies [14]. The dissociations of oJ-CgTX from sites 1 and 2 were both very slow, suggesting that the action of the toxin in the brain may be slowly reversible or irreversible as in the peripheral nervous system [9]. The binding of og-125I-CgTX was not affected by either Ca antagonists or agonist. Ca antagonists other than dihydropyridines (see Table I) bind to sites allosterically linked to the binding site of dihydropyridines [16]. The present findings indicate that ~-CgTX binds to different sites from the binding sites of any of these drugs. Further studies are required to determine whether og-CgTX and these drugs bind to distinct sites on the same molecule or to different molecules. The binding of ~-~25I-CgTX was strongly inhibited by low concentrations of divalent and trivalent metal ions. The order of the inhibitory potencies of metal ions on the binding of oJ-CgTX was very similar to that on the Ca current through the Ca channel in neuroblastoma cells that shows slow inactivation [19] and the Ca channel involved in transmitter release in nerve terminals in the brain [12]. This suggests that ~-CgTX interacts with sites related to the site of action of these metal ions in the Ca channel. Recent models [1, 7] postulate that the selective permeability of Ca 2+ through the Ca channel at high concentrations of Na ÷ and K ÷ in physiological media is achieved by preferential, high-affinity binding of two Ca ions to sites located in the pore of the channel. Divalent and trivalent metal ions may bind to the same sites, but be very poorly translocated through the channel, except in the case of Sr 2+ and Ba 2+ [6]. Thus, ~o-CgTX may bind to sites located close to the Ca2+-binding sites or allosterically related to them. After the completion of this work, the binding of to-CgTX to chick brain synaptosomes was reported [3]. Although the binding of t~-CgTX is not characterized in detail, the results are in agreement with this study in that usual Ca antagonists do not affect the binding of to-CgTX. The properties of the binding of to-CgTX reported here indicate that to-CgTX should be a useful probe for investigating the structure and function of the voltagesensitive Ca channel. We thank Dr. M. Satake for encouragement and helpful discussion. Thanks are also due to Dr. M. Okuda, Department of Psychiatry, for analysis of binding data using the L I G A N D program, kindly provided by the Biomedical Computing Technology Information Center, Nashville, TN, U.S.A. We are grateful to the drug companies (Bayer, Knoll, Tanabe and Janssen) for the supply of Ca antagonists and an agonist. This study was supported by a Grant-in-Aid (to T.A.) for Special Project Research on the Molecular Mechanisms of Bioelectrical Responses, from the Japanese Ministry of Education, Science and Culture.
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